Systematic in vitro analysis of therapy resistance in glioblastoma cell lines by integration of clonogenic survival data with multi-level molecular data

Despite intensive basic scientific, translational, and clinical efforts in the last decades, glioblastoma remains a devastating disease with a highly dismal prognosis. Apart from the implementation of temozolomide into the clinical routine, novel treatment approaches have largely failed, emphasizing the need for systematic examination of glioblastoma therapy resistance in order to identify major drivers and thus, potential vulnerabilities for therapeutic intervention. Recently, we provided proof-of-concept for the systematic identification of combined modality radiochemotherapy treatment vulnerabilities via integration of clonogenic survival data upon radio(chemo)therapy with low-density transcriptomic profiling data in a panel of established human glioblastoma cell lines. Here, we expand this approach to multiple molecular levels, including genomic copy number, spectral karyotyping, DNA methylation, and transcriptome data. Correlation of transcriptome data with inherent therapy resistance on the single gene level yielded several candidates that were so far underappreciated in this context and for which clinically approved drugs are readily available, such as the androgen receptor (AR). Gene set enrichment analyses confirmed these results, and identified additional gene sets, including reactive oxygen species detoxification, mammalian target of rapamycin complex 1 (MTORC1) signaling, and ferroptosis/autophagy-related regulatory circuits to be associated with inherent therapy resistance in glioblastoma cells. To identify pharmacologically accessible genes within those gene sets, leading edge analyses were performed yielding candidates with functions in thioredoxin/peroxiredoxin metabolism, glutathione synthesis, chaperoning of proteins, prolyl hydroxylation, proteasome function, and DNA synthesis/repair. Our study thus confirms previously nominated targets for mechanism-based multi-modal glioblastoma therapy, provides proof-of-concept for this workflow of multi-level data integration, and identifies novel candidates for which pharmacological inhibitors are readily available and whose targeting in combination with radio(chemo)therapy deserves further examination. In addition, our study also reveals that the presented workflow requires mRNA expression data, rather than genomic copy number or DNA methylation data, since no stringent correlation between these data levels could be observed. Finally, the data sets generated in the present study, including functional and multi-level molecular data of commonly used glioblastoma cell lines, represent a valuable toolbox for other researchers in the field of glioblastoma therapy resistance. Supplementary Information The online version contains supplementary material available at 10.1186/s13014-023-02241-4.


No
The second column describes the predominant clonal karyotype including all chromosomal aberrations. The third column shows whether subclones are present in the cell lines.
Supplementary Table 3: Overview of molecular glioblastoma subtype-specific chromosomal amplifications and deletions, and subtype-specific expression of relevant driver genes   [1,2].
Connections between A4GALT and glioblastoma are not reported so far.
In lung cancer models, A4GALT was shown to affect tumor progression, metastasis formation, resistance to chemotherapy, and epthelialmesenchymal transition (EMT) [3].
Connections between AP2B1 and glioblastoma are not reported so far, but have been suggested [18].
Aberrancies in expression of AP2B1 and expression of alternative splicing forms of AP2B1 were detected in different cancer entities including lung cancer and breast cancer [19,20]. NPEPPS affects the migratory and the differentation behaviour of glioblastoma cells in vitro [77].
Connections between KLHL11 and glioblastoma or other cancer entities are not reported so far.
No specific inhibitor of KLHL11 available so far.
Connections between PSMB3 and glioblastoma or other cancer entities are not reported so far.
However, the proteasome as a target for cancer therapy is of great importance [96].
No specific inhibitor of PSMB3 available so far.

None
Inhibitors of the 20S/26S core/holo-proteasome are currently trial tested in patients with different malignancies [96,97], but so far not in glioblastoma patients.
Expression of TGFBR2 correlates with expression levels of platelet derived growth factor receptor (PDFGR), a signature marker of the proneural molecular glioblastoma subtype, in glioblastoma cells [108].
No specific inhibitor of TGFBR2 available so far.
No specific inhibitor of POLR1F/TWISTNB available so far.
Overexpression of CFH in glioblastoma cells as the result of overexpression of non-metabolic indoleamine 2,3-dioxygenase 1 (IDO1) suppresses anti-tumor immune responses thereby impairing survival in syngeneic mouse models of glioblastoma [140]. Furthermore, CFH promotes the progression of glioblastoma cells by affecting AKT1 and miR-149 [141].
Finally, CFH expression is accelerated in ovarian cancer and lung cancer [142,143].

None None
Supplementary  [144]. As such, AR exhibits functions in the development and the maintenance of the reproductive system as well as in the cardiovascular, the musculoskeletal, and the haematopoietic system [145,146].
No trials in glioblastoma patients so far.
Several AR inhibitors such as Apalutamide and Darolutamide are FDAapproved for treatment of prostate cancer [171].
STAT5b has been associated with poor prognosis in glioblastoma patients [174]. Pro-malignant signaling mediated by the Epidermal Growth Factor Receptor variant III (EGFR vIII)/STAT5 axis was shown to contribute significantly to survival and migration of glioblastoma cells [175,176], and inhibition of STAT5 suppressed the proliferation, invasion, and stemness of glioblastoma cells in vitro and in vivo [177,178].
No specific inhibitor of STAT5b available so far.

TXNRD1
Thioredoxin Reductase 1 (TXNRD1) is a selenocysteinecontaining flavoenzyme of the pyridine nucleotidedisulfide oxidoreductase family. TXNRD1 is an integral component of the thioredoxin reductive system (TRX) which eliminates reactive oxygen species (ROS) thereby ensuring redox homoeostasis in cells [212,213].
TXN Thioredoxin (TXN) acts as a homodimer and is active in S-nitrosylation of cysteines, an integral step in response to intracellular nitric oxides [212,213].
Deregulation of TXN expression is reported for many cancers including glioblastoma [215,236,237]. Interference with TXN function sensitizes glioblastoma cells to radiotherapy [238].

NDUFB4
NADH:Ubiquinone oxidoreductase subunit B4 (NDUFB4) is a non-catalytic subunit of the NADH:Ubiquinone oxidoreductase enzyme complex (complex I) of the mitochondrial electron transport chain [244].
No specific inhibitor of NDUFB4 available so far.
Inhibitors of the oxidative phosphorylation (OXPHOS)related respiratory chain complex I are readily available and also trial-tested, e.g.

STK25
Serine/Threonine kinase 25 (STK25) is a member of the germinal centre kinase III (GCK III) subfamily belonging to the sterile 20 kinase superfamily. STK25 is involved in serine/threonine liver kinase B1 (LKB1) signaling, regulating neuronal polarization and morphology of the Golgi apparatus. STK25 is translocated from the Golgi apparatus to nuclei in response to anoxia, and also plays a role in the regulation of cell death [266,267].
Connections between STK25 and glioblastoma are not reported so far.
None None GSR Gluththione Disulfide Reductase (GSR) is a homodimeric flavoprotein and member of the class-I pyridine nucleotide-disulfide oxidoreductase family. GSR is a core enzyme of the antioxidant defense, as it reduces oxidized glutathione disulfide (GSSG) to its sulfhydryl form GSH [201,202].
GSR mediates drug resistance in glioblastoma cells via its function to regulate redox homeostasis [272]. GSR is deregulated in several cancer entities including lung cancer and liver cancer [273][274][275].
ABCC1 alongside with other ABC transporters is overexpressed in many cancer entities including glioblastoma [281,282]. ABCC1 contributes to therapy resistance by eliminating therapeutic agents from cancer cells. In glioblastoma, several miRNAs that target ABCC1 have been identified, and their expression levels determine the degree of therapy resistance in these tumors [283][284][285][286][287].

MK-571 [288]
Reversan [289] Thienopyrimidines [290] None Leading edge genes at the SSIR and TMZ intersection of the mammalian target of rapamycin complex 1 (mTORC1) pathway gene set for which inhibitors are readily available Name General Information Connections to Glioblastoma Inhibitors Clinical Trials PSMG1 Proteasome assembly chaperone 1 (PSMG1) dimerizes with PSMG2 to form a chaperone complex with molecular adaptor activity that is crucial for the assembly of the 20S core proteasome [291].
No specific inhibitor of PSMG1 available so far.
Bortezomib/Velcade is an FDAapproved drug for the treatment of multiple myeloma and mantle cell lymphoma [106].

PSMA4
Proteasome 20S subunit alpha 4 (PSMA4) constitutes a core subunit of the 20S core proteasome, thus playing an important role in protein homeostasis [297].
Connections between PSMA4 and glioblastoma are not reported so far.
However, polymorphisms of PSMA4 have been associated with increased susceptibility to lung cancer [298].
No specific inhibitor of PSMG1 available so far.
Bortezomib/Velcade is an FDAapproved drug for the treatment of multiple myeloma and mantle cell lymphoma [106].

HPRT1
Hypoxanthine phosphoribosyltransferase 1 (HPRT1) is an enzyme that catalyzes the conversions of hypoxanthine to inosine monophosphate and of guanine to guanosine monophosphate. Thus, HPRT1 plays a crucial role in purine salvage pathway-dependent synthesis of purine nucleotides [299,300].
Connections between HPRT1 and glioblastoma are not reported so far.
None None

SLC7A11
Solute Carrier family 7 member 11 (SLC7A11) is part of a heteromeric anionic amino acid transporter with specificity for cysteine and glutamate. In this system called Xc(-), the anionic form of cysteine is imported into cells in exchange for glutamate [319][320][321].
SLC7A11 is frequently overexpressed in glioblastoma [322], and its overexpression correlates with reduced survival and poor prognosis [308,322]. Mechanistically, overexpression of SLC7A11 increases the stem cell-like properties of glioblastoma cells [323]. Similar findings were published for other cancer entities [319,324].
No specific inhibitor of SLC7A11 available so far.

22.
Yang, S. and N.B. Hecht, Translin associated protein X is essential for cellular proliferation.